Intracellular immunization of prokaryotic cells against a bacteriotoxin.
Intracellularly expressed antibodies have been designed to bind and inactivate target molecules inside eukaryotic cells. Here we report that an antibody fragment can be used to probe the periplasmic localization of the colicin A N-terminal domain. Colicins form voltage-gated ion channels in the inner membrane of Escherichia coli. To reach their target, they bind to a receptor located on the outer membrane and then are translocated through the envelope. The N-terminal domain of colicins is involved in the translocation step and therefore is thought to interact with proteins of the translocation system. To compete with this system, a single-chain variable fragment (scFv) directed against the N-terminal domain of the colicin A was synthesized and exported into the periplasmic space of E. coli. The periplasmic scFv inhibited the lethal activity of colicin A and had no effect on the lethal activity of other colicins. Moreover, the scFv was able to specifically inactivate hybrid colicins possessing the colicin A N-terminal domain without affecting their receptor binding. Hence, the periplasmic scFv prevents the translocation of colicin A and probably its interaction with import machinery. This indicates that the N-terminal domain of the toxin is accessible in the periplasm. Moreover, we show that production of antibody fragments to interfere with a biological function can be applied to prokaryotic systems.
Chames
P
P
Laboratoire d'Ingénierie des Systèmes Macromoléculaires, Institue de Biologie Structurale et de Microbiologie du CNRS, Marseille, France.
Fieschi
J
J
Baty
D
D
Duché
D
D
D000907:Antibodies, Bacterial / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity; D000942:Antigens, Bacterial / Q000032:analysis / Q000276:immunology*; D003001:Cloning, Molecular; D003087:Colicins / Q000032:analysis / Q000276:immunology*; D003593:Cytoplasm / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D003087:Colicins; D007128:Immunoglobulin Fragments; C055966:immunoglobulin Fv; D004229:Dithiothreitol